PROJECT SUMMARY As tumors progress, cancer cells acquire characteristics that allow them to adapt to various stresses. In fact, one of the best predictors of patient outcome is disease stage at the time of diagnosis, as advanced tumors are more aggressive and difficult to treat. However, the underlying mechanisms that potentiate increased cell plasticity throughout cancer progression remain poorly understood. The ability of cancer cells to adapt has posed a particular problem for the use of targeted therapies, which are frequently rendered ineffective by the emergence of acquired resistance. The goal of this work is to elucidate molecular mechanisms that regulate the cell cycle and cell fate decisions to influence cancer progression and resistance to targeted therapy. In the F99 phase, I aim to identify novel factors that regulate the retinoblastoma (RB) pathway and influence the cellular response to inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6). CDK4/6, in complex with Cyclin D, phosphorylate and inactivate the tumor suppressor RB to drive cell cycle progression. Recently developed CDK4/6 inhibitors have shown some promise in the clinic, but every patient given these inhibitors eventually progresses, creating an urgent need to identify mechanisms of resistance. Using an in vitro genome-wide CRISPR/Cas9 screen, I recently identified loss of the E3 ligase adaptor AMBRA1 as a potential mechanism of resistance to CDK4/6 inhibition. Further, AMBRA1 loss increased Cyclin D protein stability. I hypothesize that AMBRA1, with its accompanying E3 ligase complex, targets Cyclin D for degradation, and that AMBRA1 loss could be a mechanism of resistance to CDK4/6 inhibitors in vivo. I will use molecular and biochemical assays to identify the E3 ligase that cooperates with AMBRA1 to target Cyclin D. In addition, I will combine tumor barcoding with multiplexed CRISPR/Cas9-mediated gene targeting in mouse models of non-small cell lung cancer to determine whether loss of AMBRA1 leads to CDK4/6 inhibitor resistance in vivo. In the K00 phase, I aim to elucidate the molecular mechanisms regulating cell identity in lung adenocarcinoma (LUAD). Treatment of LUAD with small molecule inhibitors targeting mutant receptor tyrosine kinases can lead to relapsed tumors that have transdifferentiated into small cell lung cancer, an aggressive neuroendocrine cancer with limited treatment options. However, the mechanism of transdifferentiation is largely unknown. I propose to develop cell line and mouse models of this transdifferentiation process in order to identify factors that regulate LUAD cell identity and ultimately identify means to prevent or reverse transdifferentiation. Together, this body of work will elucidate fundamental principles of acquired resistance and disease progression in lung cancer, which may also be applicable to other cancer types.